Cis→Trans Isomerization of Pro7 in Oxytocin Regulates Zn2+ Binding

Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn 2+ to the nine-residue peptide hormone oxytocin (OT, Cys 1 -Tyr 2 -Ile 3 -Gln 4 -Asn 5 -Cys 6 -Pro 7 -Leu 8 -Gly 9 -NH 2 , having a disulfide bond between Cys 1 and Cys 6 residues). Zn 2+ binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254 , 3899–3906 (1979)]. In the absence of Zn 2+ , we find evidence for two primary OT conformations, which arise because the Cys 6 –Pro 7 peptide bond exists in both the trans - and cis -configurations. Upon addition of Zn 2+ , we determine binding constants in water of K A = 1.43 ± 0.24 and 0.42 ± 0.12 μM −1 , for the trans - and cis -configured populations, respectively. The Zn 2+ bound form of OT, having a cross section of Ω = 235 Å 2 , has Pro 7 in the trans -configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130 , 5993–6000 (2008)], in which it was proposed that Zn 2+ binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro 7 -Leu 8 -Gly 9 -NH 2 , tail. The present work shows that the cis -configuration of OT isomerizes to the trans -configuration upon binding Zn 2+ . In this way, the proline residue regulates Zn 2+ binding to OT and, hence, is important in receptor binding. Graphical Abstract ᅟ